DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can trigger mutagenic events such as chromosome loss, deletions, duplications or translocations, events associated with tumorigenesis. Two mechanistically distinct pathways have evolved to repair DSBs: homologous recombination (HR) and non- homologous end joining (NHEJ). An alternative mechanism of NHEJ has recently been described that is independent ofthe canonical end joining factors, utilizes microhomologies (MH) to align the ends and is associated with deletions and chromosome translocations. HR initiates by the nucleolytic degradation ofthe 5' strands of DSBs to yield 3' single-stranded DNA (ssDNA) tails, a process referred to as end resection. Replication protein A (RPA) is initially bound to the ssDNA, and then displaced by Rad51 to form a nucleoprotein filament that catalyzes homologous pairing and strand invasion. Studies in budding yeast have shown that the conserved Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, together with Sae2/CtlP, initiates end resection while more extensive processing of the 5' strands requires the 5'-3' exonuclease, Exol, or the combined activities ofthe Sgsl/BLM helicase and Dna2 endonuclease. We hypothesize that the initiation of end processing by the Mre11-Rad50-Xrs2/NBS1 complex and Sae2/CtlP prevents repair by NHEJ and promotes HR; however, end resection has the potential to reveal MH internal to the break site that can be used to align ends for mutagenic MH mediated end joining (MMEJ). While HR is likely to be favored if cells form long tracts of ssDNA to assemble Rad51 nucleoprotein filaments, short ssDNA tracts might be less efficiently used for HR and directed to the MMEJ pathway. The first two aims of this project will address the role of resection initiation, in particular the role of Sae2/CtlP, extensive resection, RPA binding and assembly of the Rad51 recombinase in repair pathway choice in budding yeast. To carry out these studies we have developed novel genetic assays that will allow us to distinguish between different modes of repair. For the third aim we plan to use physical assays to monitor DNA end resection of a site-specific DSB in mouse cells to determine the domains of CtlP required for end resection, and interplay with NHEJ and 53BP1. RELEVANCE (See instructions): The repair of DNA double-strand breaks (DSBs) is essential to maintain genome integrity and to guard against cancer in humans. In this proposal, genetic and physical approaches will be used to determine how the processing of DSBs by Sae2/CtlP, and cell cycle regulation of this process, influence the pathways used to repair DSBs.